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Protein Expression in Bacterial BL21 Cells
Step 1:Transform construct into BL21 cells (if they are in another strain) * Take out and thaw 50 uL of competent Bl21 cells (in the -80 freezer, in a rack, in a box marked “60) ON ICE. **Cells are in 0.5 mL tubes with unstable tops. BRIEFLY open and close tube top immediately after taking out of the -80 and before putting on ice to release pressure. Once thawed, transfer 50 uL cells to a 1.5 mL tube. * Add 0.5 uL DNA to the 50 uL competent cells. Place on ice for 15 minutes. In the meantime, fill the 42 deg water bath and get out plates with the proper antibiotic added to warm in the incubator. * Heat shock cells, 42 deg C, 45 seconds. * Place back on ice for 2 minutes. * Add 500 uL SOC media. Shake at 37 deg C for 1-2 hours. * Plate 5-10 uL on one plate and 20 uL on the second plate. Leave in incubator to grow overnight. Step 2: * Grow up starter culture * Pick one colony from plate. Add to 5 mL culture WITH PROPER ANTIBIOTIC added. * If a frozen cell stock exists of the proper plasmid construct in BL21 cells. ' * Take out frozen cell stock. Scrape the end of a P20 pipet tip across culture to scrape up a tiny bit of cells. Release pipet tip into a 5 mL culture WITH PROPER ANTIBIOTIC ADDED.For either case…. ''Grow at 37 deg C ''overnight. * ''In addition, ''mix and autoclave proper quantity/amount of LB broth for next day’s growth. **Typically, this will be 750 mL of LB in a 2-L flask. I have been preparing 4 flasks at a time in this way. '''Step 3: Growth day On the day of growth, take out the overnight starter culture. Add a 1:1000 dilution of 1000x antibiotic (e.g., for a 750 mL culture, add 750 uL of 1000x antibiotic). TAKE OUT 1-2 mL of this LB broth and RESERVE. Then add 1:1000 dilution of the starter culture to each flask (e.g. for a 750 mL culture, add 750 uL.) Grow flasks at proper temperature. **For Drosophila proteins grow at 30 deg C, not 37. After the first two hours of growth, take the OD-600 of the cultures. Then take OD-600 every one hour until the OD-600 reaches 0.6. Induce the cultures with proper concentration of IPTG. ***The maximum FINAL IPTG concentration is 1 mM (1:1000 dilution from a 1 M stock) – however, for a new protein, never add this much IPTG. Add to final concentration of 0.2-0.5 mM IPTG.***** Drop temperature to proper growth temperature and grow for proper length of time. ***For very stable proteins, this could eventually be 18 deg C for overnight. HOWEVER, for new or unstable proteins (especially Drosophila), grow instead at 20-25 deg C for 4 hours. Ensconsin should be grown in this way. Post growth, spin down cells:'' Pour cultures into large swinging bucket containers (each will hold 750-900 mL comfortably):BALANCE in pairs!!! Spin for 20 minutes at 4000 xg, 4 deg Celsius, accel = 9, decel = 9. Pour off media from bottles. Cells should have collected on the flat bottom of the bottles. Resuspend cells into two 50-mL flasks for freezing: Working with two flasks at a time, divide 25-40 mL of PBS into the flasks. PIpet up and down and scrape bottom to resuspend the first flask. Once resuspended, transfer all liquid to the second flask; pipet up and down in the same way to resuspend the cells in the second flask. Once this has been accomplished, transfer all media into a single 50-mL conical vial. (Volume will be more than what was initially added to flasks.) Resuspend and combine the cells from the remaining two flasks in the same way. Spin down cells in final conical vials for freezing: Balance the cells in the vials. Spin again for 15-20 minutes at 4000 xg, 4 deg Celsius, accel = 9, decel = 9. Pour off media.